Cycloalkyl nitrofuryl nitrones



United States Patent Olfice 3,528,971 CYCLOALKYL NITROFURYL NITRONESRonald E. Bambury and Hyun Koo Kim, Ashland, Ohio, assignors toRichardson-Metre]! Inc., New York, N.Y., a corporation of Delaware NoDrawing. Filed July 5, 1968, Ser. No. 742,525 Int. Cl. C07d /30 US. Cl.260240 9 Claims ABSTRACT OF THE DISCLOSURE 10 Novel compounds of theformula 0..." LR. \0/ L (D) wherein: n is O or 1; A is (lower) alkylene;R is hydrogen or (lower) alkyl; and B is (a) cycloalkyl of 4 to 8 ringcarbon atoms, (b) (lower) alkylcycloalkyl wherein the cycloalkyl has 4to 8 ring carbon atoms, (c) (lower) hydroxyalkylcycloalkyl wherein thecycloalkyl has 4 to 8 ring carbon atoms, or (d) hydroxycycloalkyl of 4to 8 ring carbon atoms in the cycloalkyl group. The compounds of thisinvention have antibacterial, antifungal, and antiprotozoan activity.

This invention relates to novel cycloalkyl nitrofuryl nitrones of theabove formula which have antibacterial, antifungal and antiprotozoanactivity.

Preferably, R in the above formula is hydrogen; n is 0, i.e., A issimply a covalent bond connecting the nitrogen with the group B; and Bis unsubstituted cycloalkyl, e.g., cyclohexyl.

When n is l, the saturated aliphatic hydrocarbon group as represented byA in the above formula can be straight or branched chain divalentalkylene having from 1 to 3 carbon atoms, for example, divalentmethylene, divalent ethylene or divalent propylene.

When B of the above formula is an unsubstituted cycloalkyl group it canbe cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl.

R can be a (lower) alkyl or hydrogen. Preferably, R is hydrogen.

The term (lower) as used herein to refer to alkyl, hydroalkyl, oralkylene groups refers to such groups having from 1 to 3 carbon atoms.

Illustrative of the (lower) alkyl groups as can be represented by R or(lower) alkyls attached to a cycloalkyl group, there can be mentionedmethyl, ethyl and propyl.

Illustrative of (lower) alkylcycloalkyl groups there can be mentioned:Z-methylcyclohexyl; 3-methylcyclohexyl; 4-methylcyclohexyl;Z-ethylcyclohexyl; Z-methylcycloheptyl; 4-ethy1cyclooctyl;Z-methylcyclobutyl and 2-propylcyclopentyl.

The (lower) hydroxyalkylcycloalkyl groups can have the hydroxyalkylsubstituted in any of the positions of the cycloalkyl group. The (lower)hydroxyalkyl can be that of hydroxymethyl, hydroxyethyl orhydroxypropyl. Illustrative of the (lower) hydroxyalkylcycloalkyl groupsthere can be mentioned: 1-hydroxymethylcyclohexyl;lhydroxymethylcyclobutyl; Z-hydroxymethylcyclopentyl; 3-hydroxyethylcycloheptyl; and the like.

Illustrative of novel compounds of this invention there can bementioned: a-(5-nitro-2-furyl)-N-cyclobuty1nitrone; u- 5-nitro-2-furyl)-N-cyclopentylnitrone; a- (S-nitro-Z-furyl) -N-cyclohexylnitrone;a-(5-nitro-2-furyl)N-cycloheptylnitrone; a- (S-nitro-Z-furyl-N-cyclooctylnitrone;

3,528,971 Patented Sept. 15, 1970a-(S-nitrO-Z-furyD-N-(cyclobutylmethyl)nitrone;

zx- (5-nitro-2-furyl -N-( Z-cyclopentylethyl nitrone;

oz- (5-nitro-2-furyl -N- (cyclohexylmethyl) nitrone;

a-(S-nitro-Z-furyl)-N-(3-cycloheptylpropyl)nitrone;

OL- S-nitro-Z-furyl -u-methyl-N-cyclohexylnitrone;

a-(S-nitro-Z-furyl)-N-(Z-methylcyclohexyl)nitrone;

a- S-nitro-Z-furyl -N- 2-propylcyclohexyl nitrone;

a- 5-uitro-2-furyl -N- (4-propylcyclohexyl) nitrone;

a- (S-nitro-Z-furyl) -a-methyl-N-( l-methylcyclohexyl) nitrone;

1x-(S-nitro-Z-furyl)-N-(Z-methylcycloheptyl)nitrone;

a- 5-nitro-2-furyl -N-( l-methylcyclopentyl) nitrone;

oc- S-nitro-Z-furyl) -N- (2-hydroxycyclobutyl nitrone;

oc- 5-nitro-2-furyl -N- (2-hydroxycyclopentyl) nitrone;

oc- S-nitrcrZ-furyl) -N- (Z-hydroxycyclohexyl nitrone;

OL- (S-nitro-Z-furyl -N- (4-hydroxycyclohexyl) nitrone a-S-nitro-Z-furyl -N-( 3-hydroxycycloheptyl nitrone;

oc- 5-nitro-2-furyl) -N-[ l-hydroxycyclohexyl) methyl] nitrone;

a- S-nitro-Z-furyl) -N- [2- (Z-hydroxycyclopentyl) ethyl] nitrone;

a- S-nitro-Z-furyl -N-( 1-hydroxymethylcyclohexyl) nitrone;

a-(5-nitro-2-furyl -N- [2- (Z-hydroxyethyl) cyclobutyl] nitrone;

u- 5-nitro-2-furyl -N- [4- (Z-hydroxyethyl) cyclohexyl] nitrone; and

a- S-nitro-Z-furyl -N-[ l-hydroxycyclohexyl) methyl] nitrone.

The nitrones of this invetnion are prepared by contacting a substitutedS-nitrofuran, Formula II below, with a substituted hydroxylamine,Formula III below, to produce the nitrones of this invention as shown byFormula I and as can be illustrated by the following reaction scheme:

Formula II Formula III Formula I wherein R, (A), and B have the samemeaning as given hereinbefore, i.e., R is hydrogen or (lower) alkyl,i.e., alkyl of 1 to 3 carbon atoms, A is (lower) alkylene, (n) is 0 or1, and B is (a) cycloalkyl having 4 to 8 ring carbon atoms, (b) loweralkyl cycloalkyl wherein the cycloalkyl has 4 to 8 ring carbon atoms,(c) lower hydroxyalkylcycloalkyl wherein the cycloalkyl has 4 to 8 ringcarbon atoms, or (d) hydroxycycloalkyl having from 4 to 8 ring carbonatoms in the cycloalkyl group.

The hydroxylamine reactant of Formula III, or an acid addition saltthereof, about one-half to three molar equivalents, is mixed with onemolar equivalent of the nitrofuran reactant (Formula II) in an inertorganic solvent such as an alcohol, benzene, toluene, dioxane,chloroform, methylene chloride, carbon tetrachloride, ether and thelike, to produce the product of Formula I. The temperature of thereaction mixture is maintained at about l0 C. to +150 C. and preferablyat about 15 C. to C. for about 0.1 to 48 hours. The course of thereaction may be followed by standard analytical techniques such as gasliquid chromatography, ultraviolet absorption, thin layerchromatography, nuclear magnetic resonance, and the like, so that thereaction mixture can be worked up when the concentration of the desirednitrone reaches a maximum.

In some cases the use of a Dean-Stark type water separation apparatus isuseful in the promotion of the completion of the reaction by removal ofthe water which is formed. Alternatively, an inorganic drying agent(e.g., magnesium sulfate, sodium sulfate, etc.) or molecular sieves maybe used for the same purpose. When the acid addition salt of thehydroxyamine reactant is used, one can advantageously use a mild basesuch as sodium bicarbonate, potassium bicarbonate, sodium acetate, andthe like, to control the pH of the reaction mixture between about 4 and10.

After the reaction is complete the nitrone can be recovered from thereaction mixture by the usual techniques (filtration, evaporation of thesolvent, distillation, trituration, etc.) and can usually be furtherpurified by recrystallization.

The hydroxylamine reactant of Formula III used to prepare the nitronesof this invention can be prepared by the standard methods employed inmaking such compounds, such as reduction of the corresponding nitrocompound, reduction of oximes, hydrolysis of nitrones, oxidation ofamines, etc. Such methods are described by Peter A. S. Smith, TheChemistry of Open-Chain Organic Nitrogen Compounds, volume 11, Chapter8, published by W. A. Benjamin, Inc., New York (1966). Illustrative ofhydroxylamine reactants there can be mentioned:cyclohexylhydroxylamin'e, cyclobutylhydroxylamine, 4hydroxycyclohexylhydroxylamine, 2-hydroxycyclopentylethylhydroxylamine,Z-methylcyclohexylhydroxylamine, and4-(Z-hydroxyethyl)cyclohexylhydroxylamine.

The novel nitrones of this invention can be used for the destruction orinhibition of gram negative bacteria, gram positive bacteria, fungi, andprotozoa. In this regard, it has been found that the nitrones of thisinvention had greater activity than corresponding compounds having analiphatic alkyl instead of the cycloaliphatic group, e.g., when A B ofthe preceding Formula I is cycloheptyl, the compound exhibits activity,in the above mentioned bio logical areas, greater than the correspondingheptyl compound. For such use they can be used as disinfectantcompositions or can be administered to animals, e.g., in conventionalunit dosage forms or in the feed of veterinary animals such as chickens.

The nitrones of this invention can be employed as the activeantibacterial and antifungal agent of disinfectant compositions for thecontrol of microorganisms such as: Salmonella typhimurium; Salmonellagallinarium; Escherichia coli; Bacillus subtilis; Staphylococcus aureus;Sire ZOCOCCllS agalactiae'; Pasteurella multocida; Erysipelothrixrhusiopathiae; Candida albicans, Aspergillus fumigatus; and the like.For disinfectant use, e.g., disinfection of surgical instruments, animalwatering troughs, animal cages, etc., the nitrones of this invention canbe dispersed in an inert finely divided solid and employed as a dust inconcentrations of about 0.001% to 5%, preferably about 0.01% to 3% byweight of inert finely divided solids; or they can be dispersed in wateror oil with or without a wetting, dispering or emulsifying agent inconcentrations which can vary over a wide range such as that of about0.001% to 5%, preferably about 0.1% to 3% by weight of the liquidcomposition to prepare germicidal suspensions or emulsion which can beused, e.g., by spraying or dipping, to inhibit the growth ofmicroorganisms.

For in vivo use in the inhibition of bacteria, or protozoa, thecompounds of this invention can be administered to animals, e.g., warmblooded animals, in quantities varying from about 20 to 200 rug/kg. ofanimal weight per day. Administration can be by conventional routes suchas parenterally or orally, e.g., in tablets, capsules or in admixtureswith the animals water or feed. When admixed with animal feed or water,the concentration of the compounds of this invention can vary from about0.001% to 0.05% by weight of the animals water or feed.

Oral unit dosage forms can include tablets, capsules, powder packets,liquid suspensions, etc. Such dosages can contain from about 50 mg.(milligrams) to 500 or more mg. of the novel nitrone together withconventional carriers, e.g., lactose, corn starch, gelatin, peanut oil,and the like.

The following examples are illustrative of the invention.

EXAMPLE 1 Preparation of a-(5-nitro-2-furyl)-N-cyclohexylnitrone WL LMAnalysis.Calcd. for C H N O (percent): C, 55.45; H, 5.92; N, 11.76.Found (percent): C, 55.51; H, 5.89; N, 11.94.

EXAMPLE 2 Preparation of u-(5-nitro-2-furyl)-N-cyclopentylnitrone OzNloCH=IlI- This compound was prepared by reacting S-nitrofurfural with anequimolar amount amount of N-cyclopentylhydroxylamine as described inExample 1. Recrystallization from ether gave bright yellow crystals,(1.53 g., 68% M.P. -106" C. of the subject compound.

Analysis.Calcd. for C H N O (percent): C, 53.56; H, 5.40; N, 12.50.Found (percent): C, 53.59; H, 5.41; N, 12.44.

EXAMPLE 3 Preparation of a-(5-nitro-2-furyl)-N-cycloheptylnitrone O zNlLC H=N 0 J This compound was prepared by reacting S-nitro-furfuryl withan equimolar amount of N-cycloheptylhydroxyl amine as described inExample 1. Recrystallization from ether gave a bright yellow solid,(1.87 g., 77%), M.P. 133-135 C. of the subject compound.

Analysis.-Caled. for C H N O (percent): C, 57.13;

H, 6.39; N, 11.11. Found (percent): C, 57.06; H, 6.41; N, 11.41.

EXAMPLE 4 Preparation of a-(5-nitro-2-furyl)-N-cyclooctylnitrone Thiscompound was prepared by reacting S-nitrofurfural with an equimolaramount of N-cyclooctylhydroxylamine as described in Example 1.Recrystallization from ether gave a bright yellow solid (2.10 g., 86%),M.P. 128-130 C. of the subject compound.

Analysis.-Calcd. for C H N O (percent): C, 58.63; H, 6.81; N, 10.52.Found (percent): C, 58.58; H, 6.77; N, 10.39.

EXAMPLE 5 Preparation of a-(5-nitro-2-furyl)-N-(2-methy1-cyclohexyl)-nitrone and recrystallized from ether-hexane giving a brightyellow crystalline product of the subject compound (0.48 g., 20%), M.P.101103 C.

Analysis.Calcd. for C H N O (percent): C, 57.12; H, 6.39; N, 11.11.Found (percent): C, 57.04; H, 6.69; N, 11.08.

The N (2 methylcyclohexyl)hydroxylamine was obtained by reducing2-methylcyclohexanone oxime (25.4 g., 0.2 mole) with 270.2 ml. of 1 Msolution of borane in THF as described in Example 6. Its hydrochloridesalt was prepared by treating the hydroxylamine with etherealhydrochloric acid in an ice bath. After removal of solvent, ahygroscopic solid (23.20 g., 70%) Was obtained.

EXAMPLE 6 Preparation ofa-(5-nitro-2-furyl)-N-(2-hydroxycyclohexyl)-nitroneN-(Z-hydroxycyclohexyl)hydroxylamine hydrochloride (33.00 g., 0.197mole) in absolute ethanol (197 ml.) was stirred with S-nitrofurfural(27.70 g., 0.197 mole) in absolute ethanol (197 ml.) containing sodiumbicarbonate (16.55 g., 0.197 mole). Stirring was continued overnight andthe mixture was filtered. The filter cake was thoroughly washed withwarm ethanol until no more yellow color remained. Evaporation of thefiltrate and methanol washings gave a brown solid of the subject nitrone(2.8 g., 6%); M.P. 147-148.

Analysis.Calcd. for C H N O (percent): C, 51.96; H, 5.55; N, 11.02.Found (percent): C, 51.85; H, 5.47; N, 10.98.

The N (2 hydroxycyclohexyl)hydroxylamine hydrochloride intermediate wasprepared as follows: To 2 hydroxycyclohexanone oxime (22.60 g., 0.174mole) in THE (100 ml.) at 0 was introduced, dropwise, 470 ml. of a 1 Msolution of borane in THF, at such a rate that the temperature did notexceed 10. The reaction mixture was stirred overnight at roomtemperature, after which the temperature was lowered to 0. Next, sodiumhydroxide solution was added (24 g. of sodium hydroxide pellets in 24ml. of water) at such a rate that the temperature did not exceed 5.After refluxing for 1 hour, the reaction mixture was dried overanhydrous magnesium sulfate. The solvent was removed in vacuo to give anoil (26.5 g.), which gave a positive Tollens test at room temperature.Its hydrochloride salt was prepared by treating the hydroxylamine withethereal hydrochloric acid. After removal of solvent, a viscous whiteresidue was obtained. It was used for preparation of the nitrone withoutfurther purification.

6 EXAMPLE 7 Preparation ofa-(5-nitro-2-furyl)-N-(4-hydroxycyclohexyl)-nitrone HO gQ The subjectcompound was prepared by reacting S-nitrofurfural with an equimolaramount of N-(4- hydroxycyclohexyl)hydroxylamine hydrochloride in thepresence of sodium bicarbonate as described in Example 1.

The N (4 hydroxycyclohexyl)hydroxylamine hydrochloride intermediate wasprepared in a manner similar to that described for the preparation ofN-(Z-hydroxycyclohexyl)hydroxylamine, from 4 hydroxycyclohexanoneoxirne. Its hydrochloride salt was prepared by treating the crudehydroxylamine with ethereal hydro chloric acid. After removal ofsolvent, a viscous white residue was obtained. It was used forpreparation of the nitrone without further purification.

EXAMPLE 8 Preparation of OL- (5 -nitro-2-furyl) -N-(l-hydroxymethyl-cyclohexyl) nitrone CHzOH The subject compound wasprepared by reacting 5 nitrofurfural with an equimolar amount ofl-hydroxylaminocyclohexanemethanol oxalate in the presence of sodiumbicarbonate. Recrystallization from methanol gave a bright yellow solidof the subject nitrone.

The 1-hydroxylaminocyclohexane methanol oxalate was prepared as follows:A solution of 1-nitrocyclo hexanemethanol (15.92 g., 0.1 mole) inaqueous ethanol (75 ml. of ethanol and ml. of water) containing oxalicacid (5.40 g., 0.06 mole) was hydrogenated at room temperature and oneatmosphere pressure in the presence of 10% palladium on charcoal untilthe calculated amount of hydrogen was absorbed. The reduction mixturewas heated to boiling, filtered from the catalyst and the filtratechilled to give l-hydroxylaminocyclohexanemethanol oxalate.Recrystallization from absolute ethanol gave a pure sample.

EXAMPLE 9 Preparation of 04- 5-nitro-2-furyl) -N-[ l-hydroxycyclohexyl)-methyl] nitrone The subject compound was prepared by treating 5-nitrofurfural with an equimolar amount ofl-hydroxylaminomethyl-l-cyclohexanol oxalate in the presence of sodiumbicarbonate. Recrystallization from ethanol gave a bright yellow solidof the subject nitrone compound in 37% yield, M.P. 122124.

The l-hydroxylaminomethyl-l-cyclohexanol oxalate was prepared asfollows: A solution of l-nitrornethyl-lhydroxycyclohexane (7.16 g., 0.05mole) in aqueous ethanol (37 ml. of ethanol and 55 ml. of water)containtaining oxalic acid dihydrate (4.16 g., 0.033 mole) washydrogenated at one atmospheric pressure and room temperature in thepresence of 10% palladium on charcoal (1.0 g.) until the calculatedamount of hydrogen was ab sorbed. The reaction mixture was heated toboiling, filtered from the catalyst and the filtrate chilled to givewhite, crystalline, l-hydroxylaminomethyl-l-cyclohexanol oxalate; yield,6.70 g. (65% M.P. 144-146".

7 EXAMPLE 10 animals at the end of the test period to total in eachgroup by the abbreviation Dead/Total.

Percent level of (ED@ average test drug in the Av. mgJkg. of all levels)feed dose Dead/total (mg/kg.)

tests are shown in the following table, wherein the num- 18 bersindicate the minimum concentration of the drug in 5? millionths of agram per milliliter of contaminated sub 3-7 stance, required to inhibitthe growth of the organism. The following table shows results with andwithout the addition of the chicken serum.

SG ST SA SAG ER BS EC PM CA AF w/Broth 100 100 100 10 10 100 100 1 Serum100 100 100 100 10 10 100 100 100 10 In the above table the meanings ofthe abbreviations are as follows: SG for Salmonella gallinarium; ST forSalmonella typ himurium; SA for Staphylococcus aureus; SAG forStreptococcus agalactiae; ER for Erysipelothrix rhusiopathiae; BS forBacillus subtilis; EC for Escherichia 0011; PM for Pasteurellamultocida; CA for Candida albicans; and AF for Aspergillus fumigatws.

EXAMPLE 11 This example shows in vivo activity of a-(5-nitro-2furyl)-N-cyclohexylnitrone, also referred to as the Test Drug, byadministration of feed containing this nitrone to mice infected withSalmonella choleraesuis.

Test mice weighing 19-21 grams were used in the test. Groups of 10 ormice per group were allowed free access to feed containing variousquantities of the Test Drug from 0.1% by weight of the feed to 0.0063%.The mice are then injected intraperitoneally with 0.2 ml. of a 1:100,000 dilution of a five-hour Salmonella choleraesuis subspecieskunzendorf (ATCC #12011) brain heart infusion broth culture. Mortalityrecords were maintained for 14 days post-infection with the micereceiving the designated test feed containing the Test Drug throughoutthis period. At the completion of the test, percent survival andmilligram per kilogram dose corresponding to each level or quantity ofdrug in the feed are plotted on logarithmic probability paper in orderto determine ED (elfective dose for 50% of the animals) values. Thefeed, apart from the Test Drug, had an analysis of 24% minimum of crudeprotein, 4% minimum of crude fat, and 4.5% maximum of crude fiber. Thefeed contained: animal liver meal, fish meal, dried whey, corn and wheatflakes, ground yellow corn, ground oat groats, dehulled soybean meal,wheat germ meal, wheat middlings, cane molasses, dehydrated alfalfameal, soybean oil, brewers dried yeast, vitamin A palmitate, irradiateddried yeast (source of vitamin D riboflavin, niacin, calciumpantothenate, choline chloride. D-activated animal sterol, a-tocopherol,thiamine hydrochloride, menadione sodium bisulfite (source of vitamin Kactivity), diealcium phos phate, salt and traces of: manganous oxide,copper sulfate, iron carbonate, potassium iodate, cobalt sulfate andzinc oxide. The results of this test are shown in the following tablewherein 10 or 15 mice were used at each level (concentration). The tablealso shows the ratio of dead What is claimed is: 1. A compound of theformula wherein n is O or 1; A is (lower) alkylene; R is hydrogen or(lower) alkyl; and B is (a) cycloalkyl having 4 to 8 ring carbon atoms,(b) (lower) alkylcycloalkyl wherein the cycloalkyl has 4 to 8 ringcarbon atoms, (c) (lower) hydroxyalkylcycloalkyl wherein the cycloalkylhas 4 to 8 ring carbon atoms, or (d) hydroxycycloalkyl having from 4 to8 ring carbon atoms in the cycloalkyl group.

2. A compound of claim 1 wherein: n is 0; R is hydrogen; and B iscycloalkyl having 4 to 8 ring carbon atoms.

3. A compound of claim 2 wherein the cycloalkyl is cyclohexyl.

4. A compound of claim 1 wherein: R is hydrogen; n is 0; and B is(lower) alkylcycloalkyl wherein the cycloalkyl has 4 to 8 ring carbonatoms.

5. A compound of claim 4 wherein the cycloalkyl is cyclohexyl and thealkyl is methyl at the 2-position of said cyclohexyl group.

6. A compound of claim 1 wherein: R is hydrogen; n is 0; and B is(lower) hydroxyalkylcycloalkyl having from 4 to 8 carbon atoms in thecycloalkyl ring.

7. A compound of claim 6 wherein the cycloalkyl is cyclohexyl and thehydroxy group is in the 2- or 4-position of the cyclohexyl group.

8. A compound of claim 1 wherein R is hydrogen, n is 1 and B ishydroxycycloalkyl having 4 to 8 ring carbon atoms.

9. A compound of claim 8 wherein A is methylene, the cycloalkyl iscyclohexyl and the hydroxyl group is in the 1-position of saidcyclohexyl group.

References Cited Netherlands Published Application No. 640189, 20 pagesand 3 drawings, published I an. 5, 1965.

JOHN D. RANDOLPH, Primary Examiner US. Cl. X.R.

